Titin is multi-functional giant filamentous protein with many important effects on structure and function of muscle. The long-term goal of this project is to determine the mechanisms whereby titin influences passive and active muscle physiology, particularly with respect to the cardiovascular system. Our work in cardiac muscle has shown that titin's l-band spring region contains distinct extensible elements that shape both its short-term properties and, through alternative splicing, its long-term adaptation. These distinct regions are the tandem Ig and PEVK elements found in both cardiac and skeletal muscle and the N2B element found only in cardiac muscle. Recent evidence suggests that the PEVK and N2B elements uniquely modulate passive cardiac muscle stiffness, and that this modulation is important for shaping heart function and adaptation. We will focus on elucidating these unique mechanisms in myocardial mechanics, including the role of S100A1 in regulating the PEVK-based mechanism. We will use novel knock-out (KO) models in which the PEVK or N2B element has been excised and a model that is deficient in S100A1 (SKO). We will also critically test the hypothesis that titin regulates active myocardial force development by studying the PEVK KO and N2B KO mouse models (high passive stiffness) as well as a contrasting rat model that expresses a giant titin isoform (low passive stiffness). We will measure the length dependence of calcium sensitivity and the effect of titin on myofilament structure (using low angle X-ray diffraction). We will accomplish the proposed research using a multi-faceted approach, with experimental techniques at levels ranging across the single molecule, single cell, whole muscle, and the intact heart. Our research will contribute to understanding passive stiffness modulation, including its role in ischemia-reperfusion injury, and the interplay between titin-based passive stiffness and active force development. Passive myocardial stiffness is an important determinant of diastolic filling and utilization of the Frank-Starling mechanism, and our research will contribute to understanding the roles of titin in both diastolic and systolic dysfunction.